The NF-kB essential modulator (NEMO) is a scaffolding protein and an essential component of the of the IkB kinase (IKK) complex. Activation of the IKK complex by a number of cellular stimuli, including cytokines, bacterial and viral products and stress, is the central node of the nuclear factor kB (NF-kB) signaling pathway, which regulates cell proliferation and apoptosis, immune response and inflammation, and stress response. The IKK complex, formed by NEMO and two kinases (IKK-alpha and IKK-beta), is a primary target for drug development due to the role of NF-kB activation in the pathogenesis of autoimmune disorders, including rheumatoid arthritis, psoriasis and muscle related disorders. One approach to IKK inhibition targets the interaction between the IKK-alpha and IKK-beta kinases and NEMO. The strategy was shown to be effective utilizing a peptide, corresponding to the NEMO-binding domain of the IKKs (NBD), as an IKK complex inhibitor in in vitro and in vivo models of inflammatory arthritis, muscular dystrophy and others. The development of peptides and small molecular weight inhibitors of the NEMO-IKK interaction would greatly benefit by the knowledge of the high resolution structure of the target protein (the IKK-binding domain of NEMO). Neither X-ray nor NMR structures are available for NEMO in its unliganded state. In this proposal we aim to design and characterize novel protein constructs encompassing the IKK-binding domain of NEMO which will facilitate structural determination (aim #1) and to solve the three-dimensional structure of unliganded NEMO by NMR or X-ray crystallography (aim #2). Our multidisciplinary approach will combine protein design principles with biochemical and cellular assays to assess competence of the new constructs for IKK-binding, and biophysical studies including circular dichroism and NMR spectroscopy, to determine folding and stability of the NEMO variants. The structure of the N- terminal domain of NEMO, which encompasses the region where the IKKs bind, would allow the identification of druggable pockets for structure based design, an important step in the development of regulators of the formation of the IKK complex.